Successful cryopreservation of early mammalian embryos provides opportunities for the preservation of germplasm as well as the movement of genetics nationally and internationally. Unfortunately, the pig embryo has been more difficult than many mammalian embryos to cryopreserve. Significant advances have been made towards the successful cryopreservation of pig embryos based on the observation that pig embryos are very sensitive to hypothermic conditions and that removal of intracellular lipids (delipation) appears to alleviate this sensitivity [1-4]. Most studies have focused on in vivo produced embryos, as they are considered to be more developmentally competent than in vitro produced embryos. Alternatives to mechanical delipation include destabilizing the cytoskeleton [5] or altering the vitrification conditions [6-8].
According to prior studies on cryopreservation of in vivo produced embryos, after centrifugation of the pig oocyte or embryo with an intact zona pellucida, the polarized lipid droplets tend to remain connected with the cytoplasm of the oocyte or blastomere of the embryo via a bridge-like structure [11]. The polarized lipid droplets can redistribute back into the oocyte or blastomere during subsequent culture or cryopreservation procedures. If the perivitelline space is enlarged, the bridge-like structure will break after centrifugation and the lipid droplets will not redistribute into the cytoplasm of the oocyte or the blastomere of the embryo, but will stay within the intact zona pellucida. Thus in vivo-derived embryos need to be cryopreserved immediately after centrifugation in order to prevent lipid redistribution prior to cryopreservation [12].
Prior studies also found that the lipid droplets are abundant and large in the early stage porcine embryo and gradually decline in size and abundance as the embryo advances to and beyond the blastocyst stage [15, 16]. Interestingly, the large lipid droplets in the early stage embryos are easier to remove by centrifugation than the smaller droplets in the later stage embryos.
In-vitro production of pig embryos, such as embryos derived from in-vitro fertilization (IVF) or by nuclear transfer (NT), has been used to create disease models or potential organ donors for xenotransplantation. As a result, the demand for effective cryopreservation of in vitro produced embryos has dramatically increased. However, in-vitro produced (IVP) embryos are even more sensitive to cryopreservation, thus more difficult to cryopreserve, than their in vivo produced counterparts [9].
So far, very limited success has been achieved to cryopreserve IVP embryos. In 2006, the inventors' lab reported two litters of transgenic piglets produced from cryopreserved NT embryos [9]. Subsequently, Nagashima et al. [10] reported piglets produced from cryopreserved IVF-derived embryos. However both of these successful reports of the cryopreservation of IVF- or NT-derived embryos used mechanical delipation through centrifugation and micromanipulation [9, 10]. Mechanical delipation substantially increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation. It is also labor-intensive and time-consuming
Two other groups [13, 14] have reported the attempts to employ partial enzymatic digestion and subsequent centrifugation to improve the cryopreservation survival of pig parthenogenetic embryos and hand-made cloned embryos. Specifically, when the zona pellucida is partially digested by trypsin, pronase, or another enzyme, it swells in size, which results in an increase in the amount of space between the oocyte plasma membrane and the zona pellucida. Thus when the oocyte or embryo is centrifuged sufficient space is present for the lipids to completely separate. However, the partial enzymatic digestion treatment has some disadvantages when used for lipid separation. For example, the enzyme (such as Trypsin or Pronase) can elicit parthenogenetic activation of oocytes. Additionally, the enzymatic digestion treatment may not work consistently and needs to be observed and monitored closely in small groups, since the effect of the enzyme treatment is heavily dependent on the individual batch of enzyme. Furthermore, neither group reported any piglet produced from the cryopreserved embryos using the combination of enzymatic digestion and centrifugation method.
Therefore, there is a need to develop a practical and non-invasive method for lipid separation and cryopreservation of IVP (such as IVF-derived or NT-derived) porcine embryos, which is suitable for research and commercial purposes.